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. 2016 May 1;196(9):3525-31.
doi: 10.4049/jimmunol.1600017. Epub 2016 Mar 28.

Cutting Edge: BAFF Promotes Autoantibody Production via TACI-Dependent Activation of Transitional B Cells

Holly M Jacobs  1 , Christopher D Thouvenel  1 , Sarah Leach  1 , Tanvi Arkatkar  1 , Genita Metzler  2 , Nicole E Scharping  1 , Nikita S Kolhatkar  2 , David J Rawlings  3 , Shaun W Jackson  4
Affiliations

Cutting Edge: BAFF Promotes Autoantibody Production via TACI-Dependent Activation of Transitional B Cells

Holly M Jacobs et al. J Immunol. .

Abstract

Mice overexpressing B cell activating factor of the TNF family (BAFF) develop systemic autoimmunity characterized by class-switched anti-nuclear Abs. Transmembrane activator and CAML interactor (TACI) signals are critical for BAFF-mediated autoimmunity, but the B cell developmental subsets undergoing TACI-dependent activation in settings of excess BAFF remain unclear. We report that, although surface TACI expression is usually limited to mature B cells, excess BAFF promotes the expansion of TACI-expressing transitional B cells. TACI(+) transitional cells from BAFF-transgenic mice are characterized by an activated, cycling phenotype, and the TACI(+) cell subset is specifically enriched for autoreactivity, expresses activation-induced cytidine deaminase and T-bet, and exhibits evidence of somatic hypermutation. Consistent with a potential contribution to BAFF-mediated humoral autoimmunity, TACI(+) transitional B cells from BAFF-transgenic mice spontaneously produce class-switched autoantibodies ex vivo. These combined findings highlight a novel mechanism through which BAFF promotes humoral autoimmunity via direct, TACI-dependent activation of transitional B cells.

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Conflict of interest statement

The authors have declared that no conflict of interest exists

Figures

Figure 1
Figure 1. TACI deletion prevents BAFF-Tg autoimmunity; and excess BAFF promotes sTACI on transitional B cells
(A) Representative IgG HEp2-ANA staining. Bars, 50µm. (B) Isotype-specific anti-Sm/RNP Ab from 12-week-old WT (grey), Taci−/− (blue), BAFF-Tg (black) and Taci−/−.BAFF-Tg (red) mice. (C) Splenic B cell subset sTACI levels from wild-type (left) and BAFF-Tg (right) mice. Grey histogram: Taci−/− B cells. (D) % sTACI+ T1 and T2 B cells. (E) Taci mRNA transcript (fold change vs. WT T1/T2) from sorted WT and BAFF-Tg T1/T2 B cells (CD21lo/midCD24hi) as well as BAFF-Tg TACI+ vs. TACI− T1/T2 subsets. (F) Left panel: AA4.1 surface expression in WT (shaded) and BAFF-Tg (line) T1 B cells. Right panel: BAFF-Tg sTACI expression in AA4.1+ (blue) vs. AA4.1− (red) T1 B cells. (G) Left panel: Rag2-GFP reporter T1 and FM gating. Middle panel: Rag2-GFP histogram showing GFPneg and GFPpos gates. Right panel: Overlaid histograms of AA4.1 expression in Rag2-GFPpos T1 (red) and Rag2-GFPneg FM (grey) B cells. (H) T1 and FM gating (left), and T1 sTACI expression (right; Taci−/− B cells grey) in 12-week-old WT (upper) and Baffr−/− (lower) mice. Number equals % in TACI+ gate. Lower right panel: AA4.1 expression on Baffr−/− TACIhi T1 (red), Baffr−/− TACIlo T1 (blue) and WT FM (grey) B cells. (I) Overlaid flow plots demonstrating that TACIhi transitional cells (red) from BAFF-Tg mice are not CD138+ plasma cells (green) or CD11b+CD11c+ age-associated B cells (black; CD11c not shown). (A–I) Data representative of WT (n=16), Taci−/− (n=12), BAFF-Tg (n=15) and Taci−/−.BAFF-Tg (n=14) from ≥2 independent experiments/sorts. Error bars indicate SEM; **, P<0.01; ****, P<0.0001; by two-tailed Student’s t test.
Figure 2
Figure 2. TACIhi transitional cells exhibit an activated, cycling phenotype
(A) Overlaid flow plots from TACIlo (blue) and TACIhi (red) BAFF-Tg T1 B cells showing cell size by forward (FSC)/side scatter (SSC). (B) Left panels: Representative overlaid histograms of surface activation markers from WT (grey), TACIlo BAFF-Tg (blue) and TACIhi BAFF-Tg (red) T1 B cells. (C) Activation marker MFI in WT and BAFF-Tg T1 B cell subsets. (D) Left panel: Histogram of T1 GFP expression in Rag2-GFP (shaded) and BAFF-Tg.Rag2-GFP (line) mice. Right panel: sTACI expression in GFP+ (blue) vs. GFP− (red) T1 B cells from BAFF-Tg.Rag2-GFP mouse. (E) KREC analysis of sorted WT and BAFF-Tg T1/T2 B cells (CD21lo/midCD24hi), as well as BAFF-Tg TACIlo and TACIhi T1/T2 B cells. (A–E) Data representative of ≥2 independent experiments involving WT (n=9), BAFF-Tg (n=9), Rag2-GFP (n=5) and BAFF-Tg.Rag2-GFP (n=7) mice; error bars indicate SEM; ***, P<0.001; ****, P<0.0001, by two-tailed Student’s t test.
Figure 3
Figure 3. BCR engagement promotes sTACI, AID and T-bet expression by BAFF-Tg transitional B cells
(A) Left panel: Overlaid histograms showing Nur77-GFP expression in T1 and FM B cells from Nurr77-GFP (T1, blue; FM; grey shaded) and Nur77-GFP.BAFF-Tg (T1, red; FM; black line) mice. (B) T1 B cells were subdivided into color bins based on relative Nur77-GFP expression. Representative histograms of sTACI expression in overlaid GFP color bins from Nur77-GFP (left panel) and Nur77-GFP.BAFF-Tg (right panel) mice. (C) Representative histograms of T1 AID (left) and T-bet (right) intracellular staining in WT (shaded) and BAFF-Tg (line) mice. (D) AID levels in TACIlo (blue) vs. TACIhi (red) T1 B cells from representative BAFF-Tg mouse. (E) AID (left) and T-bet (right) expression in WT and BAFF-Tg T1 B cell subsets. (F) Aicd and Tbx21 mRNA transcript from sorted WT (□) and BAFF-Tg (■) T1/T2 B cells (fold change vs. WT). (G) Splenic immunofluorescence staining showing prominent extrafollicular IgG2c+ cells (arrow) in BAFF-Tg mice, that are absent in Taci−/−.BAFF-Tg. Bars, 100µm. (H) IgG2c (Iγ2c) mRNA transcript from sorted WT (□) and BAFF-Tg (■) T1/T2 B cells (fold change vs. WT). (A–H) Data representative of ≥2 independent experiments involving Nur77 (n=4), Nur77-GFP.BAFF-Tg (n=4), WT (n=9), BAFF-Tg (n=9); error bars indicate SEM; *, P<0.05; **, P<0.01; ****, P<0.0001, by two-tailed Student’s t test.
Figure 4
Figure 4. The cycling, TACIhi transitional cells subset is enriched for self-reactivity and spontaneously produces class-switched autoAb
(A) Rag2-GFP.BAFF-Tg T2 B cell single cell cloning. Left panels: Serial dilution curves of cloned GFPlo (n=43) and GFPhi (n=37) BCR reactivity against dsDNA, Sm/RNP and phosphorylcholine (PC) by ELISA. Right panel: AUC of reactive antibodies (defined as BCR with reactivity > threshold of 0.5 O.D.) to respective self-antigen. (B) Bar graphs showing total Ig concentrations (upper) and anti-Sm/RNP Ab (lower) in supernatants from sorted B cell subsets from WT (□) and BAFF-Tg (■) mice, cultured for 72 hours in RPMI media or stimulated with R848 (5ng/mL). Data pooled from ≥2 independent sorts. Error bars indicate SEM; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001, by two-tailed Student’s t test (A), and one-way ANOVA, followed by Tukey's multiple comparison test (B).
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